ABSTRACT Resistance to apoptosis is one of the hallmarks of cancer. Cancer cells circumvent cell death through the mitochondrial apoptosis pathway to ensure tumor growth, maintenance and resistance to current treatments. The mitochondrial apoptosis pathway is governed by the expression levels and interactions of the BCL-2 family proteins, which comprise the pro-apoptotic effector proteins BAX and BAK, the anti-apoptotic proteins e.g. BCL- 2, BCL-XL, MCL-1 and the pro-apoptotic BH3-only proteins. Cancer cells most commonly block mitochondrial apoptosis by upregulating the anti-apoptotic BCL-2 proteins that neutralize BH3-only proteins and activated BAX and BAK. Therefore, efforts have focused on the development of selective inhibitors of anti-apoptotic BCL-2 proteins to re-activate apoptosis. Inhibitors such as the FDA-approved Venetoclax rely on the function and availability of BH3-only proteins to activate BAX and BAK. However, in many tumors, BH3-only proteins can be downregulated, suppressed or deleted, making these tumors insensitive to these inhibitors and limiting their broader clinical application. We hypothesized that small-molecule direct activation of pro-apoptotic BAX via the BAX trigger site is an alternative and possibly complementary pharmacological strategy to promote apoptosis in cancer cells. This approach can promote BAX activation independently of BH3-only proteins and therefore should have the potential to overcome apoptosis blockade in resistant tumors. Our laboratory recently used unique structural and molecular insights and medicinal chemistry to develop a potent and selective compound, termed BAX Trigger Site Activator 1(BTSA1) that promotes BAX activation and induces mitochondrial dysfunction and apoptosis. Using BTSA1, we provided proof-of-concept for direct BAX activation as a therapeutic target in Acute Myeloid Leukemia and demonstrated that direct BAX activation is well tolerated in vivo. Here, we hypothesized to generate BTSA1 analogues with improved potency, oral bioavailability and pharmacokinetics. Our goal is to evaluate their activity and mechanism of action in diverse cancer models as single agents or combination treatments and investigate mechanisms of sensitivity and resistance to BAX activation and apoptosis. Moreover, we aim to identify a clinical candidate BAX activator with favorable cellular, pharmacological and safety properties. Therefore, we propose the following specific aims: 1) characterize potency, selectivity, pro-apoptotic activity, in vitro ADME/Tox and pharmacokinetic properties of BTSA1 analogues, 2) determine cellular efficacy and mechanism of action of 2nd generation BTSAs, including BTSA1.2, alone and in combination treatments using various leukemia and solid tumor cells and investigate determinants of sensitivity and resistance, 3) determine safety and therapeutic potential of select BTSAs alone or in combination therapy and investigate novel biomarkers and mechanisms of apoptosis regulation. This proposal will advance an innovative therapeutic strategy and therapeutics and inform the most suitable context for targeting BAX activation in cancer.